A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations

Hideyuki Miyashita, Shigeto Shimmura, Kazunari Higa, Satoru Yoshida, Tetsuya Kawakita, Jun Shimazaki, Kazuo Tsubota

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K15. Furthermore, only duplex feeder layers reproduced the basal K15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.

Original languageEnglish
Pages (from-to)1275-1282
Number of pages8
JournalTissue Engineering - Part A.
Volume14
Issue number7
DOIs
Publication statusPublished - 2008 Jul 1

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Keratin-15
Feeder Cells
Stem cells
Keratin
Stem Cells
Engineers
Cell culture
Population
Cells
Liquids
Keratin-3
Air
Epithelial Cells
Phenotype
3T3 Cells
Keratins
Cornea
Cultured Cells
Immunohistochemistry

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Biomedical Engineering
  • Biomaterials

Cite this

A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations. / Miyashita, Hideyuki; Shimmura, Shigeto; Higa, Kazunari; Yoshida, Satoru; Kawakita, Tetsuya; Shimazaki, Jun; Tsubota, Kazuo.

In: Tissue Engineering - Part A., Vol. 14, No. 7, 01.07.2008, p. 1275-1282.

Research output: Contribution to journalArticle

Miyashita, Hideyuki ; Shimmura, Shigeto ; Higa, Kazunari ; Yoshida, Satoru ; Kawakita, Tetsuya ; Shimazaki, Jun ; Tsubota, Kazuo. / A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations. In: Tissue Engineering - Part A. 2008 ; Vol. 14, No. 7. pp. 1275-1282.
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